Method for testing a sterilization packaging unit for its efficacy against recontamination

ABSTRACT

The invention relates to a method for testing a sterilization packaging unit provided for sterilization of objects to be sterilized, in particular medical objects to be sterilized, for its efficacy against microbial recontamination of the sterilized objects after they have been sterilized. In accordance with the invention the packaging unit is charged with nutrient medium, subjected to a sterilization process and then stored for a period of time under ambient conditions. The packaging unit is then kept under conditions in which microbes multiply for cultivation of microbes which are present in or on the nutrient medium. The packaging unit is then opened and microbes, which have multiplied in or on the nutrient medium, are observed. The method permits the efficacy of the packaging unit with respect to its recontamination protection to be tested easily and reliably.

The invention relates to a method for testing a sterilization packagingunit provided for sterilization of objects to be sterilized for itsefficacy against microbial recontamination of the sterilized objectsafter they have been sterilized. The objects to be sterilized are, inparticular, medical objects to be sterilized.

Many objects must be sterilized before they are used, particularly inthe medical field, in order to kill microbes, i.e. livingmicroorganisms, on the objects. Such medical objects to be sterilizedcan be, for example, instruments, linen or liquids. Depending on thetype of objects to be sterilized an appropriate physical or chemicalsterilization method is used. In the medical field vapor sterilizationmethods are predominantly used. These are characterized by a high levelof efficacy and environmental compatibility. In vapor sterilizationmethods sterilization is carried out with confined and saturated steam.The temperature of the steam is for example 121° C. or 134° C.Sterilization is carried out using autoclaves.

Furthermore, microbes can also be killed by high-energy ionizing beamsor by a H₂O₂-based plasma. Chemical sterilization methods includesterilization by means of ethylene oxide or formaldehyde.

Medical objects to be sterilized are packaged for sterilization. It ispossible for this purpose to use container packages, e.g. in the form ofa box, paper packages in which the objects to be sterilized are wrappedin a number of layers of paper, and other packages. The package servesto ensure the sterility achieved by the sterilization method until thesterilized objects are used.

The quality requirements for the sterilization method are high. UnderWHO guidelines and various European pharmacopoeia, out of 1 000 000“sterile” products 999 999 must be sterile. The usual sterilizationmethods are evaluated and standardized with physical-chemical andmicrobiological parameters. A known and reliable method for testing theefficacy of a sterilization method consists of subjecting a testmicrobial unit to the sterilization method to be tested, at the sametime as the objects to be sterilized. This test microbial unit is formedby a number of microbes which are particularly resistant to thesterilization process and which are enclosed as a unit by a casing.After the sterilization process this test microbial unit is opened understerile conditions and tested to see if the microbes contained thereinare capable of multiplying. If the sterilization process has taken placecorrectly no multiplication of the test microbes will take place sincethese test microbes will have been killed by the sterilization. The testmicrobes therefore represent an indication of the effectiveness of thesterilization method.

There are also quality standards for the packaging material. Thesegenerally relate to partial aspects of the packaging unit such asmaterial properties, sealing tightness of closures etc. Suitable methodsfor testing the efficacy of the package as a whole and/or of a packagingunit in use have not been developed. However, while the sterilizationmethod is regularly checked for efficacy in the manner described, forthe period of time between sterilization and the sterilized objectsbeing used, in which recontamination can take place, there are nosatisfactory methods, for example of a microbiological type, relating todetecting the efficacy of a sterilization packaging unit in preventingrecontamination of the sterilized objects, or with respect to detectingpossible recontamination of the sterilized objects.

On the one hand, the so-called microbial barrier of the packaging unitused is a factor in the recontamination of the sterilized objects. Thismicrobial barrier is the ability of the packaging unit to prevent theentry of microorganisms. On the other hand, various external influencessuch as the type of storage or transportation and mechanical stressingon the packaged sterilized objects and ambient influences such as airflow, microbial content of the air and fluctuations in air pressure arefactors in recontamination.

A known method of testing the microbial barrier quality of partialcomponents of a container package consists of generating negativepressure in the container via orifices provided in a cover of thecontainer (EN 868-1, Appendix G). By means of this negative pressure thetightness of a seal between the cover and the container body can betested. However, this is merely one specific physical method forpackages which are in the form of containers, and this method also onlyconcerns the seal between the body and the cover but not the microbialbarrier of the vapor orifices such as filters and valves. Furthermore,ambient influences, e.g. microbial content of the air, are not includedin this testing method. This method can therefore give no reliableevidence as to the extent to which recontamination of sterilized objectsin the packaging unit must be expected.

Furthermore, there is a microbiological DIN testing method (DIN 58953,Part 6) which is provided to test sterilization paper. In this method amicrobial penetration testing unit in the form of a glass laboratoryflask is closed with the sterilization paper to be tested and is testedto see whether, by cooling the air in the microbial penetration unit, anair flow passes into the glass laboratory flask and carries particlescontaining microbes through the sterilization paper when the paper hasbeen charged with spore earth prior to cooling. Provision is made toheat the glass laboratory flask to 50° C. and to cool it to 10° C.repeatedly and consecutively. Spores which may enter the glasslaboratory flask through the sterilization paper are detected andevaluated by microbiological means using incubation of the glasslaboratory flask containing nutrient medium. This testing method,however, does not relate to a packaging unit as a whole but only topaper which is used as a packaging material. This is purely a materialtest in which, apart from the application of spore earth, no furtherambient influences are included.

It is also known to test some of the sterilized objects forrecontamination after a certain period of time by means of randomsampling. However, a disadvantage of this is the fact that to do thisthe packaging unit needs to be opened and, during the test itself,microbes can reach the object being tested so that the result of thetest can thereby be rendered erroneous. However, this method is notpracticable for hospital use so that it is not routinely used inhospitals.

From the publication of by de Bruijn, A. C. P. and Kastelein, J.,Einfach-oder Mehrfach-Verpackung von Medizinprodukten:Verfahrensbewertung durch Forschung, Zentralsterilsation; 1999; 7 (5):292-303 a method is known with which the filtering effect of a packagewith respect to an aerosol consisting of 1.0 μm latex particles isdetermined by means of a particle counter (cf. loc. cit. 3., page 297,para. 1). The known method is not intended, and is not suitable fortesting packages in the form in which they are used in order todetermine their capability to maintain sterility under hospital practiceconditions. In particular, the method is not based on the determinationof the relevant end point, namely the detection of microbiologicalcontamination.

From the publication by Junghannβ U., Winterfeld S., Gabele L., KulowU., Hygienisch-mikrobiologische und technische Überprüfungen vonSterilisier-Containersystemen, Zentralsterilisation 1999; 7 (3): 154-162a method is known for testing sterilization containers in the form ofpackaging containers. In this method a test chamber is used into whichthe containers to be tested can be inserted. Before they are placed inthe test chamber the containers were fitted with the associated filtersand sterilized. The containers were then provided with solid nutrientmedia in Petri dishes and closed. A microbe-containing aerosol wasintroduced into the interior of the test chamber using a spray bottle.By means of connection fittings fitted to the containers to be testedprior to introduction into the test chamber, suction was carried outthrough the containers to be tested, by means of a hose pump. After thesuction the containers were opened again and the solid nutrient mediumdishes were removed. These were then incubated to test for microbes onthe solid nutrient medium. The colony-forming units (cfu) were thencounted.

The known method is not suited to testing an end-usage package, in theform in which it is used in practice, in order to test its barrierefficacy with respect to recontamination. It is rather the case that thecontainers need to be provided with a suction fitting before beingintroduced into the known test chamber. Furthermore, after thecontainers have been in contact with the aerosol in the test chamber,they must be removed from the test chamber and opened in order to removethe solid nutrient medium dishes and to incubate them in a separateapparatus. The container cannot therefore remain in an undisturbedcondition until the results of the test are evaluated. External sourcesof errors can therefore not be ruled out during the test. It is ratherthe case that the removal of the solid nutrient medium dishes forsubsequent incubation is associated with a risk of contamination andtherefore with the risk of imprecise measurement.

According to Junghannβ et al. only a difference value for the microbialbarrier is determined but no absolute value is obtained. In the knowntesting arrangement no evidence as to sterility could be obtainedbecause of the non-sterile handling of the solid nutrient medium.

It is therefore the object of the invention to provide a generic method,which is effective and simple to implement, for testing a sterilizationpackaging unit as a whole with respect to its efficacy againstrecontamination of the sterilization objects after they have beensterilized.

In the method in accordance with the invention the packaging unit ischarged with nutrient medium, subjected to a sterilization process andstored under ambient conditions for a period of time. The packaging unitis then subjected to conditions which promote multiplication of microbesor microorganisms in or on the nutrient medium in order to cultivateliving microbes which may be present on or in the nutrient medium. Thismay be, in particular, incubation, for example in an incubator orincubation chamber. The multiplication conditions include e.g. atemperature suitable for the multiplication of the microbes expected tobe present, and a suitable gas atmosphere. This cultivation generallytakes place over a preset period of time. The packaging unit is thenopened and microbes which have multiplied in or on the nutrient mediumare observed. If it is the case that, after the sterilization process,which is assumed to have been carried out correctly or the correctimplementation of which has been tested by means of the test microbeindicators described above, contamination of the nutrient medium byentry of microbes into the packaging unit and settlement in or on thenutrient medium has taken place, the microbes have been cultivated asdescribed above. If microbes have been found multiplying in or on thenutrient medium then this is clear evidence that microbes have enteredthe sterilization packaging unit after the sterilization process. Thismay be attributable to the fact that the sterilization packaging unithas basic faults or it may also be attributable to the fact that theambient conditions, such as humidity and the microbial content of theair, were very unfavorable in relation to the avoidance ofrecontamination.

The method in accordance with the invention is simple to implement andpermits extensive testing of a sterilization packaging unit or of afundamental construction of a sterilization packaging unit with respectto its efficacy against recontamination of the sterilized objects. Thistest is extensive because, in contrast to the prior art, it does notonly test partial functions such as the sealing tightness between thecover and the body of a specific container packaging unit, or themicrobial penetration of sterilization paper, but includes the microbialbarrier of the whole packaging unit in dependence upon material, shapeand construction features, and because the specific ambient conditionswhich influence the recontamination risk are also included in the test.In particular it is not possible with this testing method to obtainerroneous test results by opening the package.

In order to determine the influence of the ambient conditions on therisk of recontamination it is advantageous to predetermine in the mostdefined manner possible the ambient conditions and the period of storageof the packaging unit between the sterilization process and thecultivation step.

The packaging unit can be provided with objects to be sterilized as wellas with the nutrient medium. If microbes which are capable ofmultiplying are found in or on the nutrient medium after the cultivationstep it must be assumed that the sterilized objects have also beenrecontaminated in a corresponding manner. If, in contrast, the nutrientmedium, in or on which the microbes have very good to optimal livingconditions in association with the ambient conditions provided duringthe cultivation, has no multiplying microbes it can reliably be assumedthat the sterilized objects are not recontaminated. The cultivatednutrient medium therefore has an indicator function with respect torecontamination of the sterilized objects. This embodiment of the methodin accordance with the invention is therefore clearly superior to themethod of testing some of the sterilized objects which uses randomsampling and provides little definite evidence. In particular, the testresult in accordance with the method of the invention is much lessliable to error than the known random sampling test in whichcontamination of the subject of the test can take place by means of thetest process itself.

The sterilization process can take place in particular in the form ofvapor sterilization. However, other sterilization methods are alsofeasible, e.g. sterilization by means of high-energy ionizing beams orspecific chemical substances such as ethylene oxide or formaldehyde.

The nutrient medium can be a standard medium or a selective nutrientmedium which is particularly adapted to the growth or multiplication ofcertain microbes. The nutrient medium is preferably a solid nutrientmedium so that the packaging unit can be transported in a problem-freemanner with the solid nutrient medium therein. In a conventional andfavorable manner it is an agar-based solid nutrient medium. In the caseof vapor sterilization and use of an agar-based solid nutrient medium asterilization program should be selected which ensures that, subsequentto the sterilization phase, by the application of a vacuum the stillliquid agar cannot boil over because of a delay in boiling.

When using a solid nutrient medium the number of colonies grown on thesolid nutrient medium can be determined by counting as a measure of theefficacy of the packaging unit against recontamination. It is thereforealso possible to obtain quantitative evidence with respect to thebarrier effect of the packaging unit under preset ambient conditions.

The solid nutrient medium can be introduced into a thermostable glass orsynthetic material dish. The whole bottom surface of the packaging unitcan be covered by such dishes. It is also fundamentally feasible toplace the solid nutrient medium directly into a container package ifthis package has a closed bottom, i.e. in particular if it is notprovided with a valve for drainage of condensed water. Insertion ofsolid nutrient medium in dishes into the packaging unit makes the solidnutrient medium particularly simple to handle. The said covering of thebottom surface of the packaging unit with solid nutrient medium permitsa particularly high level of reliability for the method.

If, during the sterilization process, a sterilization method is usedwhich is associated with increasing the temperature of the content ofthe packaging unit then the packaging unit should be allowed to coolbefore it is stored for the period of time under the ambient conditions.When using an agar-based solid nutrient medium the packaging unit shouldcool to below 40° C. since agar is liquid at temperatures higher than40° C.

The packaging unit can be a container package, a paper package oranother type of package. In the case of the paper package it ispossible, e.g. for a sterilization screen to be used, on whichinstruments provided for sterilization are disposed.

Furthermore, provision can be made in accordance with the invention totest a sterilization packaging unit provided to sterilize objects to besterilized, in particular medical objects to be sterilized, for itsefficacy against microbial recontamination of the sterilized objectsafter they have been sterilized, in dependence upon variable ambientconditions, in that one of the methods described above is appliedrepeatedly and consecutively, wherein, however, the ambient conditionsunder which the packaging unit is stored over the period of time aredifferent upon each application of the method. A number of identicalsamples of one type of packaging unit can be simultaneously subjected tothe methods described above which differ only in respect of the ambientconditions.

This method permits the highest possible protection to be achievedagainst recontamination of sterilized objects located in a certain typeof sterilization packaging unit, in that ambient conditions are foundwhich, in association with the sterilization packaging unit, ensure thedesired protection against recontamination. In this way it is possibleto obtain evidence about the necessary hygienic quality of a chamber inwhich the packaging unit is stored, or to determine a profile ofrequirements for such a chamber. By means of the method it is thereforepossible to set ambient air hygiene characteristics for parameters suchas microbial content of the air, but also characteristics for parametersfor transport conditions such as jarring. With this embodiment of theinvention the sterilization packaging unit and also the ambientconditions are therefore parameters which should be set corresponding tothe higher aim of achieving the highest possible protection againstrecontamination or a specific level of protection while optimizing theexpense. The type of packaging unit and the ambient conditions are setand/or tested in relation to each other.

A test of this extensive type in which requirements for a chamberquality can be determined in dependence upon a particular packaging unitis permitted only by the present invention.

The invention is explained in more detail herein under with the aid ofan exemplified embodiment.

EXEMPLIFIED EMBODIMENT

A reusable sterilization container for vapor sterilizers in accordancewith EN 285 is to be tested for its efficacy in preventingrecontamination of sterilized objects. The container is a containerpackage 596×275×115 mm in size, having a cover which comprises a filter.In preparation for testing the container two open thermostable dishes(size 175×225 mm) each with 300 ml of nutrient medium CASO-agar (Merck)are placed in a screen dish (515×245×60 mm). The charged screen dish isplaced into the body of the container and the container is closed by thecover.

The container is then subjected to a sterilization process. A vaporsterilizer (autoclave) is used for this purpose. The sterilizationtemperature is 121° C., the sterilization time is 15 min. A program forsolutions which are to be sterilized is used as the sterilizationprogram. A program such as this has the advantage that the still liquidagar does not boil over because of a delay in boiling.

After the sterilization process the container is either transported andstored under the usual conditions used in practice, or the container issubjected to experimental conditions which are defined with respect tothe microbial content of the air, fluctuations in air pressure andtransport path. The duration of exposure is selected to have a value inthe range of about 1-4 days.

The container which is still closed is then placed into an incubator at37° C. for 48 hours for cultivation purposes.

After this cultivation, colonies which have grown on the solid nutrientbase are counted. These colonies are colony-forming units (cfu), thenumber of which corresponds to the number of microbes which havepenetrated by recontamination. Thus the number of colonies is a measureof the recontamination and therefore of the efficacy of the container asa barrier to ensuring sterility.

1. Method for testing a sterilization packaging unit provided forsterilization of objects to be sterilized, in particular medical objectsto be sterilized, for its efficacy against microbial recontamination ofthe sterilized objects after they have been sterilized, characterized inthat the packaging unit is charged with nutrient medium, the packagingunit is subjected to a sterilization process, the packaging unit isstored for a period of time under ambient conditions, the packaging unitis subjected to conditions in which microbes multiply for cultivation ofmicrobes which are present in or on the nutrient medium, the packagingunit is opened and microbes, which have multiplied in or on the nutrientmedium, are observed.
 2. Method as claimed in claim 1, characterized inthat when storing the packaging unit the ambient conditions and theperiod of time are preset.
 3. Method as claimed in claim 1,characterized in that prior to the sterilization process the packagingunit is charged with objects for sterilization in addition to thenutrient medium.
 4. Method as claimed in claim 1, characterized in thatvapor sterilization is used in the sterilization process.
 5. Method asclaimed in claim 1, characterized in that the nutrient medium is aselective nutrient medium.
 6. Method as claimed in claim 1,characterized in that the nutrient medium is a solid nutrient medium, inparticular an agar-based solid nutrient medium.
 7. Method as claimed inclaim 6, characterized in that the number of colonies grown on the solidnutrient medium, each of which is attributed to a microbe initiating thecolony, is determined as a measure of the efficacy of the packaging unitagainst recontamination.
 8. Method as claimed in claim 6, characterizedin that the solid nutrient medium is located in a thermostable dish madefrom glass or synthetic material.
 9. Method as claimed in claim 8,characterized in that a bottom surface of the packaging unit is coveredby dishes containing the solid nutrient medium.
 10. Method as claimed inclaim 6, characterized in that when using an agar-based solid nutrientmedium the packaging unit is allowed to cool to below 40° C. after thesterilization process, when this sterilization process is associatedwith an increase in temperature, before the packaging unit is stored forthe period of time under the ambient conditions.
 11. Method for testinga sterilization packaging unit provided for sterilization of objects tobe sterilized, in particular medical objects to be sterilized, for itsefficacy, in dependence upon variable ambient conditions, againstmicrobial recontamination of the sterilized objects after they have beensterilized, characterized in that a method in accordance with any one ofthe preceding claims is carried out repeatedly and consecutively withdifferent ambient conditions under which the packaging unit is storedover the period of time.
 12. Method as claimed in claim 2, characterizedin that prior to the sterilization process the packaging unit is chargedwith objects for sterilization in addition to the nutrient medium. 13.Method as claimed in claim 2, characterized in that vapor sterilizationis used in the sterilization process.
 14. Method as claimed in claim 3,characterized in that vapor sterilization is used in the sterilizationprocess.
 15. Method as claimed in claim 2, characterized in that thenutrient medium is a selective nutrient medium.
 16. Method as claimed inclaim 3, characterized in that the nutrient medium is a selectivenutrient medium.
 17. Method as claimed in claim 4, characterized in thatthe nutrient medium is a selective nutrient medium.
 18. Method asclaimed in claim 2, characterized in that the nutrient medium is a solidnutrient medium, in particular an agar-based solid nutrient medium. 19.Method as claimed in claim 3, characterized in that the nutrient mediumis a solid nutrient medium, in particular an agar-based solid nutrientmedium.
 20. Method as claimed in claim 4, characterized in that thenutrient medium is a solid nutrient medium, in particular an agar-basedsolid nutrient medium.